Well-known surface and extracellular antigens of pathogenic microorganisms among the immunodominant proteins of the infectious microalgae Prototheca zopfii

Microalgae of the genus Prototheca (P.) are associated with rare but severe infections (protothecosis) and represent a potential zoonotic risk. Genotype (GT) 2 of P. zopfii has been established as pathogenic agent for humans, dogs, and cattle, whereas GT1 is considered to be non-pathogenic. Since pathogenesis is poorly understood, the aim of this study was to determine immunogenic proteins and potential virulence factors of P. zopfii GT2. Therefore, 2D western blot analyses with sera and isolates of two dogs naturally infected with P. zopfii GT2 have been performed. Cross-reactivity was determined by including the type strains of P. zopfii GT2, P. zopfii GT1, and P. blaschkeae, a close relative of P. zopfii, which is known to cause subclinical forms of bovine mastitis. The sera showed a high strain-, genotype-, and species-cross- reactivity. A total of 198 immunogenic proteins have been analyzed via MALDI—TOF MS. The majority of the 86 identified proteins are intracellularly located (e.g., malate dehydrogenase, oxidoreductase, 3-dehydroquinate synthase) but some antigens and potential virulence factors, known from other pathogens, have been found (e.g., phosphomannomutase, triosephosphate isomerase). One genotype-specific antigen could be identified as heat shock protein 70 (Hsp70), a well-known antigen of eukaryotic pathogens with immunological importance when located extracellularly. Both sera were reactive to glyceraldehyde-3-phosphate-dehydrogenase of all investigated strains. This house-keeping enzyme is found to be located on the surface of several pathogens as virulence factor. Flow-cytometric analysis revealed its presence on the surface of P. blaschkeae

Prevalence of mcr-1 in E. coli from Livestock and Food in Germany, 2010–2015

Since the first description of a plasmid-mediated colistin resistance gene (mcr-1) in November 2015 multiple reports of mcr-1 positive isolates indicate a worldwide spread of this newly discovered resistance gene in Enterobacteriaceae. Although the occurrence of mcr-1 positive isolates of livestock, food, environment and human origin is well documented only few systematic studies on the prevalence of mcr-1 are available yet. Here, comprehensive data on the prevalence of mcr-1 in German livestock and food isolates are presented. Over 10.600 E. coli isolates from the national monitoring on zoonotic agents from the years 2010–2015 were screened for phenotypic colistin resistance (MIC value >2 mg/l). Of those, 505 resistant isolates were screened with a newly developed TaqMan-based real-time PCR for the presence of the mcr-1 gene. In total 402 isolates (79.8% of colistin resistant isolates) harboured the mcr-1 gene. The prevalence was depending on the food production chain. The highest prevalence was detected in the turkey food chain (10.7%), followed by broilers (5.6%). A low prevalence was determined in pigs, veal calves and laying hens. The mcr-1 was not detected in beef cattle, beef and dairy products in all years investigated. In conclusion, TaqMan based real-time PCR provides a fast and accurate tool for detection of mcr-1 gene. The overall detection rate of 3.8% for mcr-1 among all E. coli isolates tested is due to high prevalence of mcr-1 in poultry production chains. More epidemiological studies of other European countries are urgently needed to assess German prevalence data

Identification of a blaVIM-1-Carrying IncA/C2 Multiresistance Plasmid in an Escherichia coli Isolate Recovered from the German Food Chain

Within the German national monitoring of zoonotic agents, antimicrobial resistance determination also targets carbapenemase-producing (CP) Escherichia coli by selective isolation from food and livestock. In this monitoring in 2019, the CP E. coli 19-AB01133 was recovered from pork shoulder. The isolate was assigned to the phylogenetic group B1 and exhibited the multi-locus sequence-type ST5869. Molecular investigations, including whole genome sequencing, of 19-AB01133 revealed that the isolate carried the resistance genes blaVIM-1, blaSHV-5 and blaCMY-13 on a self-transmissible IncA/C2 plasmid. The plasmid was closely related to the previously described VIM-1-encoding plasmid S15FP06257_p from E. coli of pork origin in Belgium. Our results indicate an occasional spread of the blaVIM-1 gene in Enterobacteriaceae of the European pig population. Moreover, the blaVIM-1 located on an IncA/C2 plasmid supports the presumption of a new, probably human source of carbapenemase-producing Enterobacteriaceae (CPE) entering the livestock and food chain sector

Isolation Procedure for CP E. coli from Caeca Samples under Review towards an Increased Sensitivity

Due to the increasing reports of carbapenemase-producing Enterobacteriaceae (CPE) from livestock in recent years, the European Reference Laboratory for Antimicrobial Resistances (EURL-AR) provided a protocol for their recovery from caecum and meat samples. This procedure exhibited limitations for the detection of CPE with low carbapenem MIC values. Therefore, it was modified by a second, selective enrichment in lysogeny broth with cefotaxime (CTX 1 mg/L) and with meropenem (MEM 0.125 mg/L) at 37 °C under microaerophilic conditions. By Real-time PCR, these enrichments are pre-screened for the most common carbapenemase genes. Another adaptation was the use of in-house prepared MacConkey agar with MEM and MEM+CTX instead of commercial selective agar. According to the EURL-method, we achieved 100% sensitivity and specificity using the in-house media instead of commercial agar, which decreased the sensitivity to ~75%. Comparing the method with and without the second enrichment, no substantial influence on sensitivity and specificity was detected. Nevertheless, this enrichment has simplified the CPE-isolation regarding the accompanying microbiota and the separation of putative colonies. In conclusion, the sensitivity of the method can be increased with slight modifications

CTX-M-15-Producing E. coli Isolates from Food Products in Germany Are Mainly Associated with an IncF-Type Plasmid and Belong to Two Predominant Clonal E. coli Lineages

Extended-spectrum beta-lactamases (ESBL) mediating resistance to 3rd generation cephalosporins are a major public health issue. As food may be a vehicle in the spread of ESLB-producing bacteria, a study on the occurrence of cephalosporin-resistantu Escherichia coli in food was initiated. A total of 404 ESBL-producing isolates were obtained from animal-derived food samples (e.g., poultry products, pork, beef and raw milk) between 2011 and 2013. As CTX-M-15 is the most abundant enzyme in ESBL-producing E. coli causing human infections, this study focusses on E. coli isolates from food samples harboring the blaCTX-M-15 gene. The blaCTX-M-15 gene was detected in 5.2% (n = 21) of all isolates. Molecular analyses revealed a phylogenetic group A ST167 clone that was repeatedly isolated from raw milk and beef samples over a period of 6 months. The analyses indicate that spread of CTX-M-15-producing E. coli in German food samples were associated with a multireplicon IncF (FIA FIB FII) plasmid and additional antimicrobial resistance genes such as aac(6)-Ib-cr, blaOXA−1, catB3, different tet-variants as well as a class 1 integron with an aadA5/dfrA17 gene cassette. In addition, four phylogenetic group A ST410 isolates were detected. Three of them carried a chromosomal copy of the blaCTX-M-15 gene and a single isolate with the gene on a 90 kb IncF plasmid. The blaCTX-M-15 gene was always associated with the ISEcp1 element. In conclusion, CTX-M-15-producing E. coli were detected in German food samples. Among isolates of different matrices, two prominent clonal lineages, namely A-ST167 and A-ST410, were identified. These lineages may be important for the foodborne dissemination of CTX-M-15-producing E. coli in Germany. Interestingly, these clonal lineages were reported to be widely distributed and especially prevalent in isolates from humans and livestock. Transmission of CTX-M-15-harboring isolates from food-producing animals to food appears probable, as isolates obtained from livestock and food samples within the same time period exhibit comparable characteristics as compared to isolates detected from human. However, the routes and direction of transmission need further investigation

Biochemical characterization of antigenic proteins from pathogenic Prototheca species

Prototheken sind einzellige, saprophytisch lebende Algen. Obwohl sie phylogenetisch der Gruppe der Grünalgen angehören, sind diese Mikroorganismen chlorophylllos und besitzen damit einen obligat heterotrophen Stoffwechsel. Kulturmorphologisch ähneln sie Hefen der Gattung Candida, können von diesen aber mikroskopisch leicht durch die fehlende Sprossung unterschieden werden. Prototheken weisen eine asexuelle Vermehrung durch die Bildung endogener Tochterzellen auf. Medizinisch und veterinärmedizinisch sind die Mikroalgen von Bedeutung, da einige Arten Infektionen (sogenannt Protothekosen) beim Mensch und Tier hervorrufen. Dabei verfügen Prototheken über alle Voraussetzungen, die hochpathogene Erreger benötigen: eine stabile und strapazierfähige Zellwand, Widerstandfähigkeit gegenüber physikalischem, chemischem oder mechanischem Stress, Ausbildung von Dauersporen und limitierte Behandlungsmöglichkeiten. Trotz allem sind Protothekosen sehr seltene Erkrankungen. Der jeweilige Erreger sowie Verlauf und Lokalisation der Erkrankung variieren dabei zwischen den verschiedenen Wirtsspezies. Protothekosen können sowohl lokal begrenzt auftreten, häufig in Form kutaner oder subkutaner Manifestation, oder einen disseminierten Verlauf nehmen. Die zugrunde liegenden Mechanismen der Pathogenese sind dabei noch weitgehend unbekannt. Um Grundlagen zum Verständnis des Infektionsverlaufes zu schaffen, sollten in dieser Arbeit immunreaktive Proteine von Prototheca zopfii GT2 durch Western Blot Analysen detektiert und mittels MALDI-TOF MS identifiziert werden. Im ersten Abschnitt wurden hierfür Seren experimentell infizierter Kaninchen verwendet. Mit den enthaltenen Antikörpern konnten im Western Blot 24 antigene Proteine von P. zopfii GT2 detektiert werden, welche auch für die weiterführende Analyse per MALDI-TOF MS genutzt wurden. Davon gelang im Folgenden die Identifikation von 15 Proteinen. Bei diesen handelte es sich vorrangig um regulatorische Proteine und Enzyme des Stoffwechsels. Einige dieser Proteine (Malatdehydrogenase (MDH), Hitzeschockprotein 70 (Hsp70), Elongationsfaktor 1-alpha (EF-1α), 14-3-3 Protein) sind als immunreaktive Proteine anderer eukaryotischer Pathogene bekannt. Weiter wurde auch die Kreuzreaktivität der leporinen anti-P. zopfii GT2 Seren mit Proteinen von P. zopfii GT1 und P. blaschkeae getestet. Diese erwies sich als sehr hoch. 48 der kreuzreaktiven Proteine konnten ebenfalls per MALDI-TOF MS bestimmt werden. ATPase, MDH, EF-1α, Hsp70 wurden bei allen untersuchten Spezies bzw. Genotypen als antigene Proteine identifiziert. Im zweiten Abschnitt wurden analoge Untersuchungen anhand natürlicher caniner Infektionsseren durchgeführt. Auch hier konnte eine sehr starke Kreuzreaktivität verzeichnet werden. Insgesamt wurden 198 Proteinspots mittels MALDI-TOF MS analysiert. Für 86 Proteine gelang eine Identifikation. Auch hier handelte es sich zum größten Teil um Proteine der Regulation, Replikation, Proteinexpression und um enzymatische Stoffwechselproteine. Auffällig ist, dass auch hier MDH, Hsp70, EF-1α und ein 14-3-3 Protein als immunreaktive Proteine nachgewiesen wurden. Diese spielen auch eine Rolle bei Infektionen mit den eukaryotischen Erregern Anisakis sp. Schistosoma sp. und Cryptococcus sp. Außerdem wurden Glyceraldehyd-3-phosphat- Dehydrogenase (GAPDH), Triosephosphatisomerase, Enolase und Succinyl-CoA Synthetase als antigene Proteine bestimmt. Diesen sogenannten Housekeeping- Enzymen kommt bei einer Reihe von Pathogenen eine gesonderte Rolle als oberflächenassoziierte Virulenzfaktoren zu. In ersten Untersuchungen konnte in der vorliegenden Arbeit der Nachweis erbracht werden, dass GAPDH an der Zelloberfläche von P. blaschkeae exprimiert wird. Dies legt die Vermutung nahe, dass GAPDH am Infektionsgeschehen von P. blaschkeae bei der bovinen Protothekenmastitis beteiligt ist und dass P. zopfii GT2 möglicherweise über andere oberflächenassoziierte Stoffwechselenzyme verfügt. Weiterführende Untersuchungen bezüglich der Beteiligung von Enzymen am Infektionsgeschehen durch ihre Aktivität an der Zelloberfläche werden dringend benötigt.Prototheca spp. are unicellular, saprophytically living algae. Although these microorganisms belong to the phylogenetic group of the green algae, they are colorless due to the absence of chlorophyll. Therefore their metabolism is characterized as obligate heterotrophic. Macroscopically they bear a likeness to yeasts of the genus Candida but the algae can be differentiated from them easily under the microscope because they show no budding. Prototheca reproduce asexually by the formation of endogenous daughter cells. They are of medical and veterinary interest as some of the Prototheca species are associated with infections in humans and animals, known as protothecosis. They fulfill all qualifications to be highly pathogenic: a strong and resilient cell wall, resistance against physical, chemical and mechanical stress, formation of a resting cell stage and limited treatment options. However, protothecosis is a relatively rare disease. The pathogenic agent, the pathogenicity as well as the localization of the infection vary between the different host species. Protothecosis may occur as local infection for example as cutaneous manifestation but can run a disseminated course. There is little known about the underlying mechanisms of pathogenicity. To lay the basis of understanding the course of infection, the aim of this thesis was to determine immunoreactive proteins of Prototheca zopfii GT2 by western blot analysis and identify those using MALDI TOF MS. In the first part of this work, western blot analysis was used to identify immunodominant protein spots using sera of experimentally infected rabbits. With these 24 immunoreactive proteins of P. zopfii GT2 were detected, from which 15 proteins could be identified by MALDI TOF MS. Most of these proteins were proteins of regulatory functions and enzymes of the metabolism. Some of them - malate dehydrogenase (MDH), heat shock proteins 70 (Hsp70), elongation factor 1-α, (EF-1α) and 14-3-3 protein – are known as immunoreactive proteins from other eukaryotic pathogens. Further crossreactivity of the sera with proteins from P. zopfii GT1 and P. blaschkeae was tested with a high success rate. 48 of these crossreactive proteins could be determined using MALDI TOF MS, too. ATPase, MDH, EF-1α and Hsp70 were identified from western blot analysis of all tested strains. In the second part of this PhD thesis, experiments with sera from naturally infected dogs were performed as above. The canine sera display a marked crossreactivity between different species and genotypes, too. Taken together 198 proteins spots were analyzed by MALDI TOF MS from which 86 were identified. Comparable with the leporine sera, the main part of the immunoreactive proteins obtained with canine infection sera were proteins of regulation, replication, protein expression and metabolic enzymes. It is noticeable that MDH, Hsp70, EF-1α and 14-3-3 protein were also found as immunoreactive proteins. These are involved in pathogenicity of the eukaryotic pathogens Anisakis sp., Schistosoma sp. and Cryptococcus sp. Further glyceraldehyde-3-phosphate dehydrogenase (GAPDH), triosephosphate isomerase, enolase and succinyl-CoA synthetase could be determined as antigenic proteins. These so-called housekeeping enzymes are of special functions as surface-associated virulence factors in a number of pathogens. In this thesis, for the first time, the presence of GAPDH on the surface of Prototheca spp. was demonstrated using FACS analysis. It was shown that GAPDH is expressed on the surface of P. blaschkeae. This implies the assumption that GAPDH could be involved in the pathogenicity of P. blaschkeae causing bovine mastitis. It is also possible that P. zopfii GT2 disposes of other surface associated metabolic enzymes than GAPDH. It is strongly recommend that additional investigations be carried out to confirm the expression of these identified enzymes on the surface of Prototheca cells and ascertain their role in the infection process

Wissenschaftliche Begleitung des Würzburger Bildungsfonds

Wissenschaftliche Begleitung des Würzburger Bildungsfonds In Deutschland gilt der Zusammenhang zwischen Bildungserfolg und Elternhaus als besonders stark. Hier will der Würzburger Bildungsfonds ansetzen und durch finanzielle Unterstützung ausgewählter Schulen benachteiligten Kindern eine Chance auf zusätzliche Förderung ermöglichen. Die wissenschaftliche Begleitung des Würzburger Bildungsfonds zielt darauf ab, Gelingensbedingungen und bestehende Potentiale aufzudecken. Methodik und Fragestellung Die Informationen wurden durch Experteninterviews erhoben, wobei die Erfahrungen und Eindrücke der projektzuständigen Vertreter/-innen der teilnehmenden Schulen nach dem ersten Förderjahr im Fokus standen. Aufdieser Basis wurde die leitende Frage nach der besonderen Eignung des Würzburger Bildungsfonds als Fördermöglichkeit aus verschiedenen Perspektiven betrachtet. Gestaltung, Mehrwert und bestehende Potentiale waren hier ebenso von Bedeutung, wie administrative Aufgaben. Weiterhin wurde auf die Zusammenarbeit der Bürgerstiftung Würzburg und Umgebung mit den einzelnen Schulen ein besonderes Augenmerk gelegt. Mehr Chancen für Kinder – Gute Passung zwischen Konzept und Schulen Als positiv erwiesen sich mit Blick auf das erste Förderjahr die Flexibilität bzw. das hohe Maß an Eigenverantwortlichkeit und Handlungsfreiheit im Umgang mit dem unmittelbar verfügbaren Förderbudget. Den Schulen wurde über das Schuljahr hinweg die Möglichkeit gegeben, entsprechend der sehr individuellen Zusammensetzung ihrer jeweiligen Schülerschaft bedarfsgerechte Projekte zu entwickeln bzw. ihre Schüler/-innen allgemein bedarfsgerecht -und wenn nötig auch unverzüglich - zu unterstützen. Als bedeutender Aspekt erwies sich zudem die einfache administrative Handhabung. Insgesamt wurde und wird das Projekt „Würzburger Bildungsfonds“ damit als sinnvoll und wirksam in Bezug auf dessen Ziel – der Unterstützung benachteiligter Kinder – bewertet und dafür besonders geschätzt

First Detection of GES-5-Producing Escherichia coli from Livestock—An Increasing Diversity of Carbapenemases Recognized from German Pig Production

Resistance to carbapenems due to carbapenemase-producing Enterobacteriaceae (CPE) is an increasing threat to human health worldwide. In recent years, CPE could be found only sporadically from livestock, but concern rose that livestock might become a reservoir for CPE. In 2019, the first GES carbapenemase-producing Escherichia coli from livestock was detected within the German national monitoring on antimicrobial resistance. The isolate was obtained from pig feces and was phenotypically resistant to meropenem and ertapenem. The isolate harbored three successive blaGES genes encoding for GES-1, GES-5 and GES-5B in an incomplete class-I integron on a 12 kb plasmid (pEC19-AB02908; Acc. No. MT955355). The strain further encoded for virulence-associated genes typical for uropathogenic E. coli, which might hint at an increased pathogenic potential. The isolate produced the third carbapenemase detected from German livestock. The finding underlines the importance CPE monitoring and detailed characterization of new isolates

Comparison of Antimicrobial Resistances in Escherichia coli from Conventionally and Organic Farmed Poultry from Germany

In this study, resistance rates in Escherichia coli from organic and conventional poultry in Germany were compared. Isolates were randomly collected from organic and conventional broiler and turkey flocks at the farm and from turkey meat at retail. Resistance testing was performed as prescribed by Commission implementing decision 2013/652/EU. Logistic regression analyses were performed for the resistance to the different antimicrobials. Overall, resistance rates for the antimicrobials tested were lower in E. coli from organic than from conventionally raised animals. In turkeys, the percentage of isolates susceptible to all antimicrobials tested from animals and meat was twice as high from organic than from conventional origin (~50% vs. <25%). In broilers, the percentage of susceptible isolates from organic farms was five times higher than from conventional farms (70.1% vs. 13.3%) and resistance to three or more classes of antimicrobials was 1.7- to 5.0-fold more common in isolates from conventional farms. The differences between organic and conventional farming were more pronounced in broilers than in turkeys. More studies on turkeys are needed to determine whether this difference is confirmed